Mycoplasma pneumoniae is a major cause of community-acquired respiratory disease, accounting for >20% of all pneumonia and >100,000 hospitalizations annually in the U.S., and is the leading cause of pneumonia in older children and young adults. Infections are often chronic and can precipitate or exacerbate life-altering conditions including asthma and COPD, but the molecular basis for tissue tropism and persistence in the airways is not known. Mycoplasma adhesin protein P1 functions directly in receptor binding and gliding motility. Sialoglycoproteins and sulfated glycolipids such as sulfatide have been identified as receptors, but M. pneumoniae cells glide when bound to sialoglycoproteins and are static when bound to sulfatide. Our long- term goal is to understand how the dynamic interplay between M. pneumoniae and airway epithelium impacts transmission, tissue tropism, within-host spread, immune evasion, and persistence. A greater understanding of the underlying molecular recognition events and how they impact infection outcome will enhance our ability to develop new strategies to limit incidence and impact. Our central hypothesis is that M. pneumoniae adherence and gliding motility are influenced by the density and structure of airway glycans, which in turn impacts colonization pattern and within-host spread. Surprisingly little is known about the structure and distribution of airway glycans, despite their fundamental importance for diverse respiratory pathogens. Aim 1 will characterize the human airway glycome in the context of potential M. pneumoniae receptors, addressing which glycans are found where and at what levels in a human airway model and in human airway tissues. We will utilize state-of- the-art carbohydrate structural analysis to define the glycome of normal human bronchial epithelial (NHBE) cells grown in air-liquid interface culture, with which we model mycoplasma colonization. We speculate that glycan environment contributes to the distinct spatial and temporal colonization patterns observed in this model. Results from NHBE cell glycome analysis will be compared with comprehensive sulfated glycolipid analysis of human tissue samples from upper and lower airways. Aim 2 will define M. pneumoniae binding specificity and force and gliding phenotype on receptor populations, addressing which potential receptors impact binding and gliding activity. We will screen a comprehensive glycan array to determine binding specificity, determine binding strength by atomic force microscopy, and evaluate adherence and gliding on sialoglycoprotein and sulfated glycolipid receptor moieties individually and in combination at different relative concentrations. Aim 3 will characterize a subpopulation of the adhesin P1 protein that is defined by its characteristic monoclonal antibody labeling pattern, assess how changes in the abundance of this P1 subpopulation correlate with adherence and gliding function, and correlate P1 dynamics with structural changes on the terminal organelle surface, as observed by electron cryotomography.